Culture medium for tissue culture techniques

ABSTRACT

A chemically defined, protein-free culture medium embodying an anion-exchange resin and a water-soluble lipid source provides means for tissue culture.

United States Patent Torney et al.

[ June 3, 1975 CULTURE MEDIUM FOR TISSUE CULTURE TECHNIQUES Inventors:Harry L. Torney; Helen T. Torney;

Dale E. Bordt, all of Indianapolis,

Ind.

Assignee: The Dow Chemical Company,

Midland, Mich.

Filed: July 9, 1973 Appl. No.: 377,838

Related US. Application Data Continuation of Ser. No. 229,219, Feb. 24,1972, abandoned, which is a continuation of Ser. No. 58,203, July 24,1970, abandoned.

US. Cl. 195/l.7 Int. Cl Cl2k 9/00 Field of Search 195/ 1.7

OTHER PUBLICATIONS Johnson et al., J. Bacteriology, Vol. 80, pp. 406-411.

Primary ExaminerRichard L. Huff Attorney, Agent, or Firm-Maynard R.Johnson [5 7] ABSTRACT A chemically defined, protein-free culture mediumembodying an anion-exchange resin and a watersoluble lipid sourceprovides means for tissue culture.

3 Claims, N0 Drawings CULTURE MEDIUM FOR TISSUE CULTURE TECHNIQUESCROSS-REFERENCE TO RELATED APPLICATION This is a continuation ofapplication Ser. No. 229,219, filed Feb. 24, 1972, and now abandonedwhich is a cont. of Ser. No. 58,203, filed July 24, 1970, now abandoned.

BACKGROUND OF THE INVENTION A number of viruses are known to be ofeconomic importance by reason of their role as the causative agent ofcertain diseases in animals. In the investigation of such viruses, andin development of vaccines and other materials for prophylaxis for suchdiseases, it has been a common practice to culture living animal cellsin vitro. Many viruses, as well as various economically andscientifically significant microorganisms such as bacteria, rickettsiaeand pleuropneumonia-like organisms (PPLO) have been made susceptible tolaboratory examination by virtue of propagation in tissue culture ofliving animal cells. For example, many viruses have been isolated fromdiseased animals, propagated, and modified by attenuation or killed byspecialized techniques after propagation by means including tissueculture as a central element. Tissue culture techniques are employed inthe production of vaccines, in assay of viruses, in antibody assay, ininterferon assay, in virus isolation procedures, in propagation ofestablished virus strains, and a number of other techniques. Widespreaduse of tissue culture techniques has has given economic significance tovarious cell lines, propagated in vitro for several successivetransfers, which are employed in assay procedures, genetic studies,vaccine production and a number of other areas.

Many media for the propagation and maintenance of cells in tissueculture are known. Among these are a number of chemically-defined media,such as medium 199 of Morgan, Morton and Parker, Proc. Soc. Exptl. Biol.& Med. 7311-8 (1950), Eagles Basal Medium, Science 122:50l504 (1955),Science, 123:845847 (1956), J. Biol. Chem. 226:l9l206 (1957) and EaglesMinimum Essential Medium, Science, 130:432437 (1959) which may alsoinclude various balanced salt solutions (BSS) such as Hanks BSS, EarleBSS, Dulbecco Phosphate-buffered saline, Puck Saline F., Puck Saline Gand the like. Merchant et al., Handbook of Cell and Organ Culture,Burgess Publishing Co., Minneapolis (1964). Such media materials consistof defined identifiable carbohydrates, minerals, amino acids, vitamins,salts, etc., in definite amounts, thus ensuring the identity of themedium from batch to batch. Other materials employed in tissue culturetechniques, usually together with a chemically defined medium orbalanced salt solution, include natural extracts and proteinhydrolysates such as yeast extract, lactalbumin hydrolysate, Scherermaintenance medium, tryptone, tryptose, peptone, La Ye (lactalbuminhydrolysate extract) and the like. Such other extract and hydrolysatematerials are not chemically defined, and are thus not susceptible of aswide application as are the synthetic chemically defined media. Both thechemically defined media and the natural extract and protein hydrolysatematerials can be sterilized by autoclaving prior to use. A third classof material utilized in tissue culture techniques, often as a supplementto a chemically defined medium, are the natural animal protein materialssuch as albumins, globulins, or animal serum such as human, calf,bovine, lamb, rabbit serum or the like. The natural materials arecharacteristically neither chemically defined nor chemically pure. Theycan carry viral, rickettsial, PPLO, bacterial or other biologicalcontaminants, and cannot be sterilized by heat without thermaldegradation or inactivation of the desired animal protein. They may alsobe cytotoxic as respect some tissue, or they may carry undesired viralinhibitors or materials unsuitable for use in vaccine production orassay tech niques.

Some of the problems inherent in the use of serum in virus productioncan be alleviated in many cases. For example, serumcontaining growthmedia employed in growing cells for virus propagation can be replacedwith a serum-free medium prior to inoculation with the virus to bepropagated. However, the replacement of the serum-containing mediumtypically requires repeated washing of the cells to ensure removal ofthe serum. The increased handling during the washings increases risks ofcontamination. Further, elaborate precautions must be taken to minimizethe risk of contamination, thus making the multiple washing operation atedious and expensive procedure. Propagation of the cells in anautoclavable serum-free or protein-free medium would eliminate the needfor such washings, as well as eliminating the risks and expense incurredin using serum in the growth medium. It would thus be desirable toprovide a serum-free medium which can be used in both tissue culturegrowth and virus propagation.

Further, it is desirable to avoid the use of proteins and the likenatural products since such materials are subject to considerablevariability from batch to batch. Various autoclavable media for thegrowth of animal cells and for propagation of viruses have beenemployed. However, in most cases much more rapid and abundantpropagation of cells and of viruses is obtained when such media aresupplemented with animal serum or albumin. A chemically defined mediumfree of animal protein which provides enhanced cell and viruspropagation would clearly be desirable.

A serum-free chemically defined medium comprising a buffered aqueoussolution of carbohydrates, amino acids, mineral salts and vitamins, awater-soluble lipid source and a basic anion-exchange resin is describedby Torney, U.S. Pat. Application Ser. No. 725,517 filed Apr. 30, 1968.The medium is taught to be useful in growing Leptospira organisms.

SUMMARY OF THE INVENTION In accordance with the present invention thereis provided an improved protein'free composition suitable for tissueculture of animal cells, and for production of viruses with excellentyields. The improved composition comprises a conventionalchemically-defined culture medium including a buffered aqueous solutionof sugars, amino acids, mineral salts and vitamins and further comprisesa growth-enhancing amount of watersoluble lipid source and an amount ofa basic anionexchange resin. The invention further comprises methods foremploying the improved compositions in tissue culture and in propagationof viruses.

In carrying out the invention the medium may be prepared directly bydissolving together in distilled water each of the required ingredientsand adding to the resulting solution the required growth-enhancingamount of ion exchange resin and the water-soluble lipid source.Alternatively in the latter step the lipid source and ion exchange resinmay be mixed together separately, with heating if desired, and addedtogether to the solution of other essential ingredients. In practice, itis convenient to supply the essential amino acids, sugars, salts andvitamins primarily in the form of one of the standard chemically definedmedia such as Medium 199 Eagles Basal Medium, Eagles Minimum EssentialMedium, or the like.

In preparing the compositions of the invention it is critical andessential to employ a growth-enhancing amount of each of the lipidsource and the anionexchange resin. The exact growth-enhancing amount oflipid source and anion-exchange resin to be employed in a given case isdependent upon such factors as the particular chemically defined mediumemployed, the precise lipid source and anion-exchange resin employed,the tissue employed, whether the cells to be grown are primary cells orfrom a cell line, whether the cells to be grown have been previouslypropagated in protein-free media, and the like. In a particular case,the optimum amounts of lipid source and anionexchange resin to beemployed can be ascertained by conventional rangefinding procedures.Good results are obtained when employing from about 0.5 to about 4 partsby volume of the resin and from about 0.01 to about 0.1 parts by volumeof water-soluble lipid source per 100 parts by volume of the finishedmedium. Excellent results have been obtained with an improved mediumcontaining from about 0.1 to about 2 parts by volume of resin and fromabout 0.002 to about 0.004 parts by volume of lipid source per 100 partsof a conventional chemically-defined standard medium such as medium 199or Eagles Basal Medium. Suitable anionexchange resins include weaklybasic anion-exchange resins and strongly basic anion-exchange resins.Typical weakly basic anion-exchange resins may be prepared in accordancewith the teachings of U.S. Pat. Nos. 2,591,574; 2,597,439 and 2,642,417.In such operations a vinyl-aromatic compound such as styrene iscopolymerized with a minor proportion of a crosslinking agent such asdivinylbenzene to produce a cross-linked polystyrene. The latter is thenreacted in accordance with the teachings of the above patents with ahalomethylating agent such as chloromethyl methyl ether to produce ahalomethylated polystyrene. This halomethylated polymeric product isthereupon reacted with a primary or secondary amine or with a polyaminesuch as a polyalkylenepolyamine to produce the desired weakly basicanion-exchange resin. In preparing a strongly basic anion-exchange resinthe halomethylated polymeric product, prepared in the above manner, isreacted with a tertiary amine to introduce a plurality of quaternaryammonium groups on the polymer structure and thereby produce the desiredanion-exchange resin. Commercially available resins may be employed as,for example, the weakly basic anion-exchange resin sold by the Rohm &Haas Company under the trademark Amberlite IR-45 or the strongly basicanion-exchange resins sold by The Dow Chemical Company under thetrademarks Dowex l and Dowex 2. In general, it is preferred to employ aresin having a high water-holding capacity indicating a relatively lowdegree of crosslinking, for example, a resin prepared by initiallycopolymerizing styrene with only about 2 to 4 percent of divinylbenzene.It is further preferred to employ such a resin having a particle size offrom about 20 mesh to about 400 mesh.

Any suitable water-soluble lipid source may be employed, such as, forexample, sodium oleate or a mixture of sodium oleate and sodiumstearate. In a preferred embodiment, however, the lipid requirement ofthe culture medium is supplied in the form of a polysorbate, that is, awater soluble polyoxyethylene derivative of a sorbitan monoester of afatty acid. Such polysorbates are available commercially from the AtlasPowder Company under the trademark Tween. Of such polysorbates thepolyoxyethylene derivative of sorbitan mono-oleate (polysorbate 80) andparticularly the polyoxyethylene derivatives of sorbitan monostearate(polysorbate 60) are preferred.

In employing the medium, conventional tissue culture techniques may beused. The usual precautions to avoid contamination are necessary. Goodresults have been obtained when the sugar, amino acid, salt and vitaminsolution of the medium of the invention, after sterile filtration, isinoculated with living animal cells, the required amounts of resin andlipid source are added, after autoclaving, and the resulting culturesthereafter maintained at a temperature of 28 to 37 C. for a period oftime until the desired growth of the tissue is obtained. Inoculation ofthe resulting cell sheet with virus, addition of fresh medium,harvesting and virus purification and assay are similarly carried outaccording to conventional techniques.

The growth of cells and formation of a tissue monolayer in the use ofthe medium of the invention is generally accompanied by growth of cellson particles of the anion-exchange resin, particularly when theinoculated medium is incubated in a stationary culture vessel. In otherchemically defined media or in media containing serum, but lacking theresin and lipid source of the present invention, growth of a tissuemonolayer is confined to the surfaces of the culture vessel whichcontact the medium. In the present medium, growth also can take place onthe resin particles, which, being insoluble, rapidly settle in theculture vessel, forming a bed or layer of particles on the lowersurfaces thereof.

In the case of primary cells, growth typically occurs both on the resinparticles as well as on the walls of the culture vessel. When the cellsemployed are from a cell line which has been previously adapted to growin a medium containing animal serum or other animal protein, tissuegrowth has been observed to be on the resin particles to the exclusionof the walls of the culture vessel.

When virus is propagated in tissue culture utilizing the resin and lipidsource-containing medium, improved virus titers can be generallyobtained as compared either with other chemically-defined media or withmedia containing animal protein. In addition, significant increases invirus yield have been obtained by employing increased volumes of mediumand inoculum. In utilizing such increased volumes of material,three-fold or greater increases in virus titers have been observed withtwo-fold increases in thevolume of medium, and inoculum employed, theconcentrations of resin, lipid source, sugars, amino acids, salts,vitamins,

initial cell dispersion and virus inoculum being the same. Thus, thepresent invention provides further improvements in propagation ofviruses in addition to that which would result from the improvements intissue culture resulting from the use of the invention.

In certain cases, typically in tissue culture of cells from a celllineadapted to growth in serum-containing media, it can be desirable toemploy an autoclaved extract such as yeast extract or an autoclavedprotein hydrolysate in conjunction with the chemically-defined medium ofthe invention. While the resulting medium is not chemically defined, itis nevertheless free of unsterilized components such as serum. Inaccordance with the invention, an autoclaved natural product such asyeast extract, lactalbumin hydrolysate, peptone, peptose, tryptone ortryptose is employed in an amount sufficient to enhance the growth ofthe cell line cells adapted to grow in serum-containing media in thesterile resin and lipid source-containing medium, and such cells arepropagated in such autoclaved, sterile medium for a sufficient number ofserial transfers to adapt the cell line cells to the absence of serum.In general, adaptation of the cell line to the absence of serum, asindicated by tissue growth in the sterile, autoclaved medium comparableto growth of cells of the same origin in serum-containing media, isobtained after from 1 to 2 or 3 or 4 passages in the serum-free medium.The cells can then be further adapted to the sterile, chemically definedmedium of the invention by carrying out additional serial transfers withdecreasing amounts of autoclaved natural product until good tissuegrowth is obtained in the absence of the autoclaved natural prod uct.Conventional procedures, such as terminal dilution techniques, can beemployed to facilitate adaption of the originally serum-dependent cellline to the chemically-defined medium of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS The following examples illustratethe invention but are not to be construed as limiting the same.

EXAMPLE 1 A Lipid Concentrate is prepared by dissolving polysorbate 60(Tween 60) in glass-distilled water to give a solution containing 500milligrams of polysorbate 60 per 100 milliliters of solution.

The Resin Ingredient is similarly prepared in bulk .prior to formulationof the medium of the invention by forming a bed of resin in a glasscylinder and washing the resin with the following materials in the orderindicated by the numerals below:

The washed resin is then dispersed in aqueous 0.1 Normal sodium chloridesolution, between and 50 grams of resin to 100 milliliters of solution.The pH of the dispersion is adjusted to pH 7.0 by the addition ofaqueous 1 Normal sodium hydroxide with constant agitation. The resin issterilized by autoclaving with about five volumes of water. The anionexchange employed is a strongly basic anion-exchange resin (Dowex 1)obtained by reacting trimethylamine with a halomethylated polymer isbead form. The halomethylated polymer is prepared by reactingchloromethyl methyl ether in the presence of a zinc chloride catalystwith beads of a copolymer of 98 percent of styrene and 2 percent ofdivinylbenzene.

EXAMPLE 2 Medium 199 of Morgan, Morton and Parker, a synthetic nutrientmedium, is prepared, sterile filtered, inoculated with primary chickembryo cells at a cell concentration of 630,000 cells per milliliter,and placed in each of four culture vellels A, B, C, D, in the amount ofmilliliters per vessel. 0.0025 milliliters of lipid source, polysorbate60 (Tween 60) are added to two such vessels, A and B. The lipid sourcebeing added in the form of an autoclaved Lipid Concentrate of Example l,0.5 milliliters of the Lipid Concentrate being employed to provide0.0025 milliliter of lipid source. 0.3 milliliter of anion exchangeresin is added to vessels A and C, the resin being previously sterilizedby autoclaving. The culture vessels are incubated at a temperature of 35C. Tissue growth is evaluated over a seven day period, beginning 48hours after inoculation. Poor growth is observed in vessel D, goodgrowth is observed in both vessels B and C with formation of a good cellsheet on the glass culture vessel surface in contact with the medium,and good growth is observed in vessel A, the growth being poor on theglass but good on the resin particles.

EXAMPLE 3 The procedure of Example 2 is repeated employing variousamounts of the polysorbate 6O lipid source and of the resin, prepared asdescribed in Example 1. 75 milliliters of Medium 199 with 3.75milliliters of calf serum is employed for comparison. Tissue isincubated at a temperature of 35 C. Beginning 24 hours after incubation,the extent of cell growth is evaluated daily for seven days. The extentof growth is recorded on a scale from 0, indicating no growth to 4,indicating excellent growth with formation of an extensive cellmonolayer. The results are set out in the following table.

Employing procedures similar to those described in the precedingExamples, medium 199, and the polysorbate 60 and resin of Example 1 areemployed in the growth of measles virus (live attentiated measles virus,Schwarz strain) in chick embryo tissue culture by the procedure ofSchwarz, US. Pat. No. 3,133,861. The culture vessels are observed forcell growth, and the virus is harvested by conventional techniques andtitered in VERO cells. The amounts of resin, polysorbate 60, calf serumand medium 199 employed in each ves- The following Examples illustratethe use of the sel are set out below in milliliters (ml) together withthe method and medium of the invention in propagation of resultsobtained. cells from a cell line.

*Tissue Yield of Measles Appearance Virus in TCID Vessel ResinPolysorbate 6O Calf Serum Medium 199 after 7 days 0.2 Milliliters**Glass Resin A 0 0 l ()0 4 None 1259 B 0 (l 3.75 100 4 None 316 C 0.60.0025 0 100 4 Good 1995 D 1.2 0.0050 0 200 4 Good 7943 *Cellsinoeulated with measles virus one day after incubation 01' cells.Cytopathic effects in chick embryo cell shecl indicative of virusreplication is observed in vessels C and D about l-Z days prior toobservation of cytopathie effects in vessels A and B.

Tissue Culture Infecting Dose-50 percent in 0.2 milliliter of harvestliquid as titcred in VERO cell culture.

EXAMPLE The procedure of Example 4 is repeated employing chick embryotissue culture for determination of virus 2 yield and employing variousamounts of medium. The results are set out below.

Amount of Ingredient in Milliliters Virus Yield TCID I Mumps virus(Jones strain) is grown in chick embryo tissue culture using the mediumof the invention, with medium 199 with and without added calf serumemployed for comparison. In the first such operation, medium 199containing about cells per milliliter is employed with a mumps virusinoculum of one milliliter of a preparation containing lO' TCID livemumps virus. In the second operation, medium 199 containing about 10cells per milliliter is employed with a mumps virus inoculum of 10 TCIDThe virus yield, as determined by titration in chick embryo tissueculture, is set out below.

Ingredient in Milliliters Virus Yield EXAMPLE 7 Cells from theMadin-Darby Bovine Kidney cell line (MDBK) are dispersed in Eaglesmedium which has been previously sterilized by filtration. The celldispersion is aseptically measured into sterile Blake bottles, 150milliliters of the dispersion being added to each bottle. 7.5milliliters of fetal lamb serum are added to one of the bottles (A): 1.2milliliters of resin and 0.002 milliliters of polysorbate 60 (0.4milliliters of Lipid Concentrate) prepared as described in Example 1 areadded to three other Blake bottles, B, C and D, the

resin and polysorbate 60 having been sterilized by autoclaving prior touse. In addition, 15 milliliters of an aqueous 5 percent by weightsolution of lactalbumin hydrolysate, sterilized by autoclaving, areadded to bottle C, and 0.75 grams of autoclaved tryptone are added tobottle D. A fifth bottle, E, is employed as a check,

containing only the cell dispersion in Eagles medium. The bottles areincubated at a temperature of 35 C. for 10 days, after which the extentof tissue growth is evaluated. Tissue growth is found to be very poor inthe check bottle E wherein only Eagles medium has been employed, andexcellent cell growth is observed in bottle A, containing added lambserum. No cell growth is observed on the glass in bottles B, C and D;however, moderate to heavy tissue growth is observed on the resinparticles. Tissue growth is rated 2, 4 and 3 in bottles B, C, and D,respectively, employing a rating system from O for no growth to 4 forexcellent growth.

Resin Tween Calf Serum Me- TCID,-, ,/O.2 Milliliters dium Operation 1Operation II The results obtained in the preceding Examples 2-6 6indicate the improvements in both cell and virus yields which can beobtained by the practice of the present invention. Further, such resultsindicate the outstand- EXAMPLE 8 Bottles B and C of Example 7 areincubated for an additional day at which time tissue growth is heavy onthe resin particles in both bottles. The cells have these g improvementswhich can be Obtained y utilization bottles are subcultured into freshmedium identical to of the medium of the invention in increased ratiosof volume to surface area.

that employed in the respective bottle in Example 7. MDBK cells ofbottle D are subcultured after 17 days.

In the subculturing procedure employed, the supernatant liquid isdecanted, leaving the cell sheet and resin in the bottle. Cells areharvested by addition of 15 milliliters of a sterile ATV solution(aqueous trypsin and Versene EDTA chelating agent), incubation at 35 C.for 15 minutes followed by agitation and decantation of liquidcontaining released cells. The process is repeated employing lmilliliters of ATV solution and 10 minutes incubation, after which cellsremaining on the resin are removed by rinsing with Eagles medium. TheATV suspensions and Eagles medium rinse are combined, filtered throughsterile gauze, and centrifuged to separate the cells. The cells areresuspended in fresh Eagles medium and a cell concentration of about100,000 cells per milliliter for subculturing in the same procedureemployed in Example 7. Excellent cell growth is observed early in theincubation of the third subcultures (fourth passage in the medium B, Cor D) with notably increased growth as compared to the original passagesof Example 7.

EXAMPLE 9 In a procedure similar to that employed in Examples 7 and 8,MDBK cells previously grown for several serial passages in mediacontaining serum are dispersed in filtered Eagles medium for aconcentration of about 100,000 cells per milliliter. The dispersion isplaced in Blake bottles, 150 milliliters per bottle, and theanionexchange resin of Example 1 and polysorbate 60, and varying amountof sterile tryptone, tryptose or lactalbumin hydrolysate are added. Thebottles are incubated for 14 days before evaluation of tissue growth.The added ingredients and the growth observations are set out below.

Ingredient in Milliliters Ingredient in Grams minerals employed in theculture medium can be varied considerably without departing from theconcept of the present invention provided that the essential components,namely a growth-enhancing amount of a watersoluble lipid source andanion-exchange resin, are incorporated in the medium.

What is claimed is:

1. In a tissue culture method comprising the steps of inoculating aprotein-free, sterile tissue culture medium with living animal tissuecells and incubating said inoculated tissue culture medium underconditions conductive to cell propagation, the improvement whichcomprises the steps of mixing a growth-enhancing amount of awater-soluble lipid source selected from the group consisting ofpolysorbate 60 and polysorbate 80 with said medium, in an amount of fromabout 0.0010 to about 0.100 part by volume of lipid source per 100 partsby volume of medium, intimately contacting said medium with agrowth-enhancing amount of a strongly basic anion-exhance resin obtainedby reacting trimethylamine with a halomethylated polymer prepared byreacting chloromethyl methyl ether in the presence of a zinc chloridecatalyst with beads of a copolymer of about 98 percent of styrene and 2percent of divinylbenzene and thereafter incubating said tissue culturemedium, cells and polysorbate lipid source in intimate contact with saidstrongly basic anion-exchange resin under conditions conducive to cellpropagation for a time sufficient to obtain substantial propagation ofsaid cells.

2. The method of claim 1 wherein the lipid source is polysorbate 60.

Tissue Growth* After 14 Days Aqueous 5'7: Resin Polysorbate 80lactalbumin Tryptone Tryptose Hydrolysute Very Poor 1.5 0.002 0.75 Verygood 1 .5 0.002 5.0 075 Excellent 1.5 0.002 075 Good *Tissuc growth isobserved to be on the resin particles and not on the glass.

3. The method of claim 1 wherein the lipid source is polysorbate 80.

1. IN A TISSUE CULTURE METHOD COMPRISING THE STEPS OF INOCULATING APROTEIN-FREE, STERILE TISSUE CULTURE MEDIUM WITH LIVING ANIMAL TISSUECELLS AND INCUBATING SAID INOCULATED TISSUE CULTURE MEDIUM UNDERCONDITIONS CONDUCTIVE TO CELL PROPAGATION, THE IMPROVEMENT WHICHCOMPRISES THE STEPS OF MIXING A GROWTH-ENHANCING AMOUNT OF AWATER-SOLUBLE LIPID SOURCE SELECTED FROM THE GROUP CONSISTING OFPOLYSORBATE 60 AND POLYSORBATE 80 WITH SAID MEDIUM, IN AN AMOUNT OF FROMABOUT 0.0010 TO ABOUT 0.100 PART BY VOLUME OF LIPID SOURCE PER 100 PARTSBY VOLUME OF MEDIUM, INTIMATELY CONTACTING SAID MEDIUM WITH AGROWTH-ENHANCING AMOUNT OF A STRONGLY BASIC ANION-EXHANCE RESIN OBTAINEDBY REACTING TRIMETHYLAMINE WITH A HALOMETHYLATED POLYMER PREPARED BYREACTING CHLOROMETHYL METHYL ETHER IN THE PRESENCE OF A ZINC CHLORIDECATALYST WITH BEADS OF A COPOLYMER OF ABOUT 98 PERCENT OF STYRENE AND 2PERCENT OF DIVINYLBENZENE AND THEREAFTER INCUBATING SAID TIS SUE CULTUREMEDIUM, CELLS AND POLYSORBATE LIPID SOURCE IN INTIMATE CONTACT WITH SAIDSTRONGLY BASIC ANION-EXCHANGE RESIN UNDER CONDITIONS CONDUCTIVE TO CELLPROPAGATION FOR A TIME SUFFICIENT TO OBTAIN SUBSTANTIAL PROGATION OFSAID CELLS.
 1. In a tissue culture method comprising the steps ofinoculating a protein-free, sterile tissue culture medium with livinganimal tissue cells and incubating said inoculated tissue culture mediumunder conditions conductive to cell propagation, the improvement whichcomprises the steps of mixing a growth-enhancing amount of awater-soluble lipid source selected from the group consisting ofpolysorbate 60 and polysorbate 80 with said medium, in an amount of fromabout 0.0010 to about 0.100 part by volume of lipid source per 100 partsby volume of medium, intimately contacting said medium with agrowth-enhancing amount of a strongly basic anion-exhance resin obtainedby reacting trimethylamine with a halomethylated polymer prepared byreacting chloromethyl methyl ether in the presence of a zinc chloridecatalyst with beads of a copolymer of about 98 percent of styrene and 2percent of divinylbenzene and thereafter incubating said tissue culturemedium, cells and polysorbate lipid source in intimate contact with saidstrongly basic anion-exchange resin under conditions conducive to cellpropagation for a time sufficient to obtain substantial propagation ofsaid cells.
 2. The method of claim 1 wherein the lipid source ispolysorbate
 60. 80.